Method for determining viable cell count

ABSTRACT

A method for conveniently and quickly determining the viable cell count and/or survival ratio of cells. This method comprises measuring the fluorescence intensity of a test sample subjected to a treatment with a fluorescent probe for nucleic acid capable of exclusively staining dead cells and the fluorescence intensity of the test sample subjected to not only the treatment with the above-mentioned probe but also another treatment of damaging cell membrane and them comparing these intensities.

TECHNICAL FIELD

[0001] The present invention provides a method for conveniently andquickly determining the number of viable cells (i.e., viable cell countor viable cell number) and/or cell viability (i.e., the survival rate ofcells) by subjecting cells to a treatment with a fluorescent probe fornucleic acid, capable of exclusively staining dead cells, and also to atreatment of damaging cell membrane, which is applicable for assessmentof cytotoxicity in the field of research and development forpharmaceuticals, agricultural chemicals, cosmetics, foods, etc., thefield of research for medicine, pharmacy and biology and the field ofclinical test and diagnosis.

BACKGROUND ART

[0002] Pharmaceuticals, agricultural chemicals, cosmetics, foods, etc.are often assessed for their safety, pharmaceutical and medicinaleffect, etc., relying on cytotoxicity. For an index for thecytotoxicity, viable cells are often counted. For detecting and assayingthe viable cell number or cell viability, the following prior techniqueshave been proposed: (1) Trypan Blue staining technique; (2) MTT assaysystem (Journal of Immunological Methods, Vol. 65, pp. 55-63, 1983); (3)a method described in U.S. Pat. No. 5,314,805; (4) a method described inMolecular Medicine, Vol. 32, pp. 1340-1342, 1995; (5) a method describedin U.S. Pat. No. 5,534,416; etc.

[0003] In the technique (1), cells stained with Trypan Blue which stainsexclusively dead cells in blue color are visually counted with the nakedeye in a field under a microscope. In the system (2), MTT is a substratefor mitochondrial dehydrogenase in cells and the amount of bluish purpleformazan which is produced as a product of said enzymatic reaction isassayed by a spectrophotometer. In the method (3), a single sample ismade to react with two kinds of fluorescent probes (Calcein AM andethidium homodimer) and then fluorescence of both viable cells and deadcells is simultaneously measured whereby the viable cell number isdetermined. Although Calcein AM is a substance which usually generatesalmost no fluorescence, it is decomposed by the action of esterase inviable cells to produce calcein emitting strong fluorescence. Ethidiumhomodimer is a fluorescent probe for nucleic acid, having a propertycapable of passing through damaged cell membranes only, of specificallyinvading into dead cells only and of binding to nucleic acids. Thefluorescence of ethidium binding to nucleic acids is about 40-foldgreater than that of free ethidium homodimer. Through those measuringmethods, it is possible to easily and quickly determine the cytotoxicityby pharmaceuticals, etc. on multiplates. In the method (4), the cellviability is determined via (a) measuring (i) the fluorescence intensityof test samples treated with propidium iodide which stains dead cell DNAonly and (ii) the fluorescence intensity of test samples treated withHoechst 33258 which stains not only dead cell DNA but also viable cellDNA and (b) comparing both intensities. In the method (5), the cellviability is determined via (a) measuring (i) the fluorescence intensityof test samples treated with a cyanine fluorescent dye (Dye I)(disclosed in U.S. Pat. No. 5,436,134) which stains not only dead cellDNA but also viable cell DNA and (ii) the fluorescence intensity of testsamples treated with either a cyanine fluorescent dye (disclosed in U.S.Pat. No. 5,321,130) or a dyeing agent emitting fluorescence upon actingon viable cells (both agents are called “Dye II”) and (b) comparing bothintensities.

[0004] However, in the technique (1), since it is difficult to judgewhether they are Trypan Blue stains or not and hard to measure themunless visual observation is applied by the naked eye, it takes muchtime to complete the measurement. In the system (2) and method (3), bothare principally relying on an enzymatic reaction and, therefore, thoseare apt to be affected by temperature and pH during the measurement,enzymatic reaction time and variations in enzymatic activity among cellstrains. Further, in the system (2), not only a centrifugal operationbut also an operation of dissolving hardly soluble formazan formed as aresult of the enzymatic reaction is necessary. In the method (3), thereare disadvantages that (i) blood serum esterase in a cell culture mediumdecomposes calsein AM and, therefore, the serum is to be removed bywashing the cells for several times but, since dead cells are apt to beremoved by washing, precise measurement is not achieved unless a systemusing serum-free cell culture medium is applied; and that (ii) themeasurement is complicated because two kinds of fluorescence aremeasured. In addition, in the methods (4) and (5), there aredisadvantages that (i) the operation is complicated because two kinds offluorescence are to be measured, (ii) when cell viability is measured bytreating one sample with two kinds of fluorescent dyes, the fluorescentdye used later is badly affected by that used earlier, and (iii) inorder to determine the cell viability (survival ratio), it is necessaryto prepare a calibration curve for each measurement.

DISCLOSURE OF THE INVENTION

[0005] The present inventors have conducted various investigations inorder to provide a method for determining viable cell number whichsatisfies the conditions including: (1) viable cell numbers in a largenumber of samples can be precisely determined in a convenient and quickmanner; (2) detectable for even test samples where blood serum coexistsin the cell culture medium; (3) free of troublesome operations to thesample, such as cell washing, centrifugal separation of cells,dissolution of crystals, etc.; (4) the measured result is rarelyaffected by variations depending on temperature for the measurement, pHfor the measurement, time for reacting with a reagent, kinds of cell,etc. As a result thereof, they have succeeded in accomplishing thepresent invention.

[0006] The present invention relates to:

[0007] (I). a method for determining the number of viable cells (i.e.,viable cell number) and/or cell viability (i.e., the survival ratio ofcells), which comprises

[0008] (i) measuring the fluorescence intensity of a test samplesubjected to a treatment with a fluorescent probe (or dye) for nucleicacid, said probe being capable of exclusively staining dead cells,

[0009] (ii) measuring the fluorescence intensity of a test samplesubjected to (a) a treatment with said fluorescent probe for nucleicacid and (b) a treatment of damaging cell membranes, and

[0010] (iii) comparing both fluorescence intensities; and

[0011] (II). a kit for the measurement of viable cell number and/or cellviability by the following steps:

[0012] (i) measuring the fluorescence intensity of a test samplesubjected to a treatment with a fluorescent probe for nucleic acid,capable of exclusively staining dead cells,

[0013] (ii) measuring the fluorescence intensity of a test samplesubjected to treatments with (a) said fluorescent probe for nucleic acidand (b) an agent capable of damaging cell membranes, and

[0014] (iii) comparing both fluorescence intensities, which comprises

[0015] (A) said fluorescent probe for nucleic acid, capable ofexclusively staining dead cells, and

[0016] (B) said agent capable of damaging cell membranes.

[0017] One aspect of the present invention is:

[0018] (1). a method for determining viable cell number and/or cellviability, which comprises

[0019] (i) measuring the fluorescence intensity of a test samplesubjected to a treatment with a fluorescent probe for nucleic acid,capable of exclusively staining dead cells,

[0020] (ii) measuring the fluorescence intensity of a test samplesubjected to not only (a) a treatment with said fluorescent probe fornucleic acid but also (b) a treatment of damaging cell membranes, and

[0021] (iii) comparing both fluorescence intensities;

[0022] (2). the method according to the above (1) wherein

[0023] (i) the test sample is treated with a fluorescent probe fornucleic acid, capable of exclusively staining dead cells, and theresulted fluorescence intensity is measured, and

[0024] (ii) said test sample is then subjected to a treatment fordamaging cell membranes, so that cells would become extinct, therebyintensifying the fluorescence thereof and the resultant fluorescenceintensity thereof is measured;

[0025] (3). the method according to the above (1) wherein

[0026] (i) the test sample is treated with a fluorescent probe fornucleic acid, capable of exclusively staining dead cells, and theresulted fluorescence intensity of said test sample is measured, and

[0027] (ii) another test sample is subjected to not only (a) a treatmentwith said fluorescent probe for nucleic acid but also (b) a treatment ofdamaging cell membrane, and the resulted fluorescence intensity thereofis measured;

[0028] (4). the method according to any of the above (1) to (3) whereinthe fluorescent probe for nucleic acid is a cationic fluorescent stain(or dye) for nucleic acid;

[0029] (5). the method according to any of the above (1) to (4) whereinthe cell membrane is damaged with an agent capable of damaging cellmembranes;

[0030] (6). the method according to the above (5) wherein the agentcapable of damaging cell membranes is a surface-active agent;

[0031] (7). the method according to any of the above (1) to (6) whereineach operation in a series of the following steps is conducted by anautomated apparatus or system:

[0032] (i) measuring the fluorescence intensity of a test samplesubjected to a treatment with a fluorescent probe for nucleic acid,capable of exclusively staining dead cells,

[0033] (ii) measuring the fluorescence intensity of a test samplesubjected to not only (a) a treatment with said fluorescent probe fornucleic acid but also (b) a treatment of damaging cell membranes, and

[0034] (iii) comparing both fluorescence intensities;

[0035] (8). a kit for determining viable cell number and/or cellviability by the following steps:

[0036] (i) measuring the fluorescence intensity of a test samplesubjected to a treatment with a fluorescent probe for nucleic acid,capable of exclusively staining dead cells,

[0037] (ii) measuring the fluorescence intensity of a test samplesubjected to treatments with (a) said fluorescent probe for nucleic acidand (b) an agent capable of damaging cell membranes, and

[0038] (iii) comparing both fluorescence intensities, which comprises

[0039] (A) said fluorescent probe for nucleic acid, capable ofexclusively staining dead cells, and

[0040] (B) said agent capable of damaging cell membranes.

[0041] Another aspect of the present invention is:

[0042] (9). the method according to any of the above (1) to (3) whereinthe fluorescent probe for nucleic acid is ethidium homodimer or acyanine fluorescent dye capable of exclusively staining dead cells;

[0043] (10). the method according to any of the above (1) to (3) whereinthe fluorescent probe for nucleic acid is a cyanine fluorescent dyecapable of exclusively staining dead cells;

[0044] (11). the method according to any of the above (5), (6), (9) and(10) wherein the agent capable of damaging cell membranes is apolyoxyethylene nonionic surface-active agent or alcohol nonionicsurface-active agent;

[0045] (12). the method according to any of the above (5), (6), (9) and(10) wherein the agent capable of damaging cell membranes is at leastone surface-active agent selected from the group consisting of TritonX-100 (trade name), Triton X-114 (trade name) and Nonidet P-40 (tradename);

[0046] (13). the kit according to the above (8) wherein the fluorescentprobe for nucleic acid is a cationic fluorescent stain for nucleic acid;

[0047] (14). the kit according to the above (8) wherein the fluorescentprobe for nucleic acid is ethidium homodimer or a a cyanine fluorescentdye capable of exclusively staining dead cells;

[0048] (15). the kit according to the above (8) wherein the fluorescentprobe for nucleic acid is a cyanine fluorescent dye capable ofexclusively staining dead cells;

[0049] (16). the kit according to any of the above (8), (13), (14) and(15) wherein the agent capable of damaging cell membranes is apolyoxyethylene nonionic surface-active agent or alcohol nonionicsurface-active agent; and

[0050] (17). the kit according to any of the above (8), (13), (14) and(15) wherein the agent capable of damaging cell membranes is at leastone surface-active agent selected from the group consisting of TritonX-100 (trade name), Triton X-114 (trade name) and Nonidet P-40 (tradename).

[0051] Still another aspect of the present invention is:

[0052] (18). an apparatus (or system) for determining viable cell numberand/or cell viability, which comprises at least

[0053] (a) a means for supplying a test sample with a fluorescent probefor nucleic acid, capable of exclusively staining dead cells,

[0054] (b) a means for measuring the fluorescence intensity of the testsample which is treated with said fluorescent probe for nucleic acid,

[0055] (c) a means enabling us to damage cell membrane,

[0056] (d) a means for measuring the fluorescence intensity of the testsample which is subjected to not only (A) a treatment with saidfluorescent probe for nucleic acid but also (B) a treatment of damagingcell membrane, and

[0057] (e) a means for comparing the fluorescence intensity (b) with thefluorescence intensity (d);

[0058] (19). the apparatus according to the above (18) wherein (i) thetest sample to be measured is treated with a fluorescent probe fornucleic acid which stains dead cells only and the intensity for theemitted fluorescence resulted thereby is measured and then (ii) saidtest sample is subjected to a treatment of damaging the cell membranethereof, so that the cells are destined to become extinct and theintensity for the fluorescence intensified thereby is measured;

[0059] (20). the apparatus according to the above (18) or (19) whereinthe above-mentioned (i) and (ii) are conducted in an automated manner;

[0060] (21). the apparatus according to the above (20) wherein eachtreatment is conducted in an automatically controlled manner by the useof a microprocessor according to a program which is installed in advancetherein;

[0061] (22). the apparatus according to any of the above (18) to (21)wherein said apparatus is equipped with

[0062] (a) a device for holding an agent capable of damaging cellmembranes,

[0063] (b) a device for holding a fluorescent probe for nucleic acidcapable of exclusively staining dead cells,

[0064] (c) an injector for drug capable of supplying the test samplewith the agent from the above-mentioned device (a) or

[0065] (d) a container, such as a microplate or cuvette for testequipped with a mixer, capable of holding the test sample for assaytherein, and

[0066] (e) a spectrofluorophotometer; and

[0067] (23). the apparatus according to (18) wherein the apparatus isequipped with

[0068] (a) a device capable of, for a single test sample,

[0069] (i) measuring the intensity for the fluorescence emitted by thetest sample which is subjected to a treatment with a fluorescent probefor nucleic acid, capable of exclusively staining dead cells, and

[0070] (ii) then measuring the intensity for the fluorescenceintensified by a treatment of damaging cell membranes wherein the cellswould become extinct, or

[0071] (b) a device capable of

[0072] (i) measuring the intensity for the fluorescence emitted by atest sample subjected to a treatment with the fluorescent probe fornucleic acid, and

[0073] (ii) measuring the intensity for the fluorescence emitted byanother test sample subjected to (A) a treatment with the fluorescentprobe for nucleic acid and (B) a treatment of damaging cell membranes.

[0074] Still another aspect of the present invention is:

[0075] (24). a method for evaluating the toxicity of a substance, whichcomprises

[0076] (a) treating cells with the substance requested to be evaluatedfor toxicity,

[0077] (b) treating the resulting test sample with a fluorescent probefor nucleic acid, capable of exclusively staining dead cells, followedby measuring the intensity for the fluorescence emitted by the resultanttest sample,

[0078] (c) subjecting the resulting test sample to a treatment with thefluorescent probe for nucleic acid as well as a treatment of damagingcell membranes, followed by measuring the intensity for the fluorescenceemitted by the resultant test sample,

[0079] (d) determining viable cell number and/or cell viability viacomparing both fluorescence intensities;

[0080] (25). the method according to the above (24) wherein

[0081] (i) cells are treated with a substance which is to evaluated fortoxicity,

[0082] (ii) the resulting test sample to be measured is treated with afluorescent probe for nucleic acid, capable of exclusively staining deadcells, followed by measurement of the fluorescence intensity of theresulting test sample and

[0083] (iii) this test sample is subjected to a treatment of damagingcell membrane, so that the cells would become extinct, followed bymeasurement of the intensity for the fluorescence intensified thereby;and

[0084] (26). the method according to the above (24) wherein

[0085] (i) cells are treated with a substance which is to evaluated fortoxicity,

[0086] (ii) one test sample to be measured is treated with a fluorescentprobe for nucleic acid, capable of exclusively staining dead cells,followed by measurement of the fluorescence intensity of the resultingtest sample and

[0087] (iii) another test sample is subjected to (a) a treatment withthe fluorescent probe for nucleic acid as well as (b) a treatment ofdamaging cell membrane, followed by measurement of the intensity for thefluorescence emitted by the resultant test sample.

BRIEF DESCRIPTION OF DRAWINGS

[0088]FIG. 1 is an embodiment of an apparatus (or system) fordetermining viable cell number and/or cell viability according to thepresent invention. In this apparatus, the present invention can beconducted by counting dead cells on a single test sample, followed bycounting total cells.

[0089] 1 . . . . . . agent capable of damaging cell membranes

[0090] 2 . . . . . . injector for drug

[0091] 3 . . . . . . dye (for nucleic acid) capable of staining deadcells

[0092] 10 . . . . . . light source

[0093] 6 . . . . . . cuvette (for test sample) equipped with a mixer

[0094] 5, 9 and 11 . . . . . . lens

[0095] 7 . . . . . . transducer capable of transforming analog signalinto digital

[0096] 4 and 12 . . . . . . polarizing plate

[0097] 13 . . . . . . photomultiplier

[0098] 8 . . . . . . control system

[0099] 14 . . . . . . excitation spectroscope

[0100] 15 . . . . . . fluorescence spectrophotometer

[0101]FIG. 2 is another embodiment of an apparatus (or system) fordetermining viable cell number and/or cell viability according to thepresent invention. In this system, the present invention can beconducted by preparing several test samples to be measured under asingle condition, counting dead cells for one half of said test samplesby the above-mentioned method while another half thereof is subjected toa treatment with fluorescent probe for nucleic acid and also to atreatment of damaging cell membrane, followed by, similarly to the caseof the above method, measuring the number of dead cells for the testsamples for counting dead cells and determining the total number ofcells.

[0102] 1 . . . . . . diluted cell suspension

[0103] 2 . . . . . . agent capable of damaging cell membrane

[0104] 3 . . . . . . dispenser

[0105] 4 . . . . . . dye (for nucleic acid) capable of staining deadcells

[0106] 5 . . . . . . 96-well microplate equipped with a plate mixer

[0107] 6 and 13 . . . . . . polarizing plate

[0108] 7, 10 and 12 . . . . . . lens

[0109] 8 . . . . . . transducer capable of transforming analog signalinto digital

[0110] 9 . . . . . . control system

[0111] 11 . . . . . . light source

[0112] 15 . . . . . . excitation spectroscope

[0113] 16 . . . . . . fluorescence spectrophotometer

[0114] 14 . . . . . . photomultiplier

[0115]FIG. 3 shows a relation between the number of floating cells andthe fluorescence intensity measured by the present invention. It isshown that there is a proportional relationship between the number ofcells and the fluorescence intensity.

[0116]FIG. 4 shows a relation between the number of adhesive cells andthe fluorescence intensity measured by the present invention. It isshown that there is a proportional relationship between the number ofcells and the fluorescence intensity.

BEST MODE FOR CONDUCTING THE INVENTION

[0117] The test sample to be measured to which the present invention isapplicable may be any cell so far as it has no cell wall. Examples ofsuch cells are animal cells, a part of microbial cells, protoplastsderived from microorganisms and plants wherein cell membranes areremoved and the like. Any animal cells are used as test samples.Examples thereof include animal body fluid cells (blood cells,lymphocytes, etc.) and animal cancer cells (animal tumor cells). Thosesamples may be anything so far as they are under an environment wherethe cell can be alive. Examples thereof are cells in a cultured medium.When pharmaceuticals, agricultural chemicals, cosmetics, foods, etc.having cytotoxicity are made coexisting in the living environment ofthose samples, a part or all of the cells in the sample become deadcells. When viable cell numbers are determined under such anenvironment, assessment of the cytotoxicity can be achieved.

[0118] The fluorescent probe for nucleic acid capable of exclusivelystaining dead cells (or fluorescent probe for nucleic acid which stainsdead cells only) means the agent having the following characteristicfeatures:

[0119] (1) it passes through damaged cell membrane but does not passthrough undamaged cell membrane;

[0120] (2) it emits weak fluorescence or hardly emits the fluorescencein a state where it is not bonded to nucleic acid; and

[0121] (3) it emits strong fluorescence when bonded to nucleic acid.

[0122] Embodiments of the fluorescent probe for nucleic acid may includecationic fluorescent stains (dyes) for nucleic acid, such as ethidiumhomodimer and a cyanine fluorescent dye; ethidium halides such asethidium bromide; propidium halides such as propidium iodide; and thelike. It is preferred that ethidium homodimer or a cyanine fluorescentdye capable of exclusively staining dead cells is used in the presentinvention. It is particularly preferred that a cyanine fluorescent dyewhich can exclusively stain dead cells is used herein. The cyaninefluorescent dyes capable of exclusively staining dead cells includethose described in U.S. Pat. No. 5,321,130. Examples of the cyaninefluorescent dye useful in the present invention are BOBO-1 Iodide,BOBO-3 Iodide, BO-PRO-1 Iodide, BO-PRO-3 Iodide, POPO-1 Iodide, POPO-3Iodide, PO-PRO-1 Iodide, PO-PRO-3 Iodide, TOTO-1 Iodide, TOTO-3 Iodide,TO-PRO-1 Iodide, TO-PRO-3 Iodide, YOYO-1 Iodide, YOYO-3 Iodide, YO-PRO-1Iodide, YO-PRO-3 Iodide (all of them are trade names; manufactured byMolecular Probes, U.S.A.) and the like. Fluorescent emission wavelengthvaries depending upon variations in measuring conditions such as kindsof cells to be assayed and species of targets which are to be assessedfor cytotoxicity wherein said targets may include pharmaceuticals,agricultural chemicals, cosmetics and foods. Since each fluorescentprobe for nucleic acid has its own intrinsic excitation wavelength andfluorescent emission wavelength, an appropriate fluorescent probe fornucleic acid may be suitably selected and used depending upon themeasuring condition. Further, there are some cases where the fluorescentprobe for nucleic acid itself has its own fluorescence in addition tothe culture medium and the target to be assessed for cytotoxicity, it isnecessary to measure the fluorescence intensity (Fb) of the backgroundderived thereby with a fluorometer in advance.

[0123] The above-mentioned fluorescent probe for nucleic acid has aproperty that it passes through damaged cell membranes or the cellmembrane of dead cells but is unable to pass through undamaged cellmembranes or the cell membrane of viable cells. Therefore, when thisfluorescent probe is applied to a test sample in which both viable anddead cells are coexisting, the fluorescent probe selectively permeatesinto the dead cells and bonds with nucleic acids to emit a strongfluorescence. When this fluorescence intensity (Fd′) is measured by afluorometer, the dead cell numbers can be determined. To be morespecific, fluorescence intensity (Fd) corresponding to the number ofdead cells is calculated from the following equation:

Fd=Fd′−Fb

[0124] From the resulting value, the number of dead cells can bedetermined by means of a calibration curve or the like. It is alsopossible to directly determine the cell viability without calculatingthe number of dead cells.

[0125] The methods for damaging cell membrane for the test sample to bemeasured include a method wherein an agent capable of damaging cellmembrane (or agent for damaging cell membrane) is applied, a methodwherein cell membranes are damaged (or injured) by a physical means suchas application of ultrasonic wave, etc.

[0126] The agent capable of damaging cell membranes (i.e., agent fordamaging cell membranes) may be any agent as long as it damages orinjures the cell membrane of viable cells with the result that all theviable cells in the system would (or are destined to) become extinct (orwould be killed). Examples of the agent capable of damaging cellmembranes are surface-active agents; acids such as hydrochloric acid andsulfuric acid; bases such as sodium hydroxide and potassium hydroxide;etc. In order to distinctly conduct the measurement of the fluorescenceintensity which will be mentioned later, it is preferred to use asurface-active agent among the above-mentioned ones. More specificexamples thereof are polyoxyethylene ether nonionic surface-activeagents such as Triton X-100, Triton X-114, Triton X-305, Triton X-405,Brij-35, Brij-56 and Brij-58 (all of them are trade names; manufacturedby Pierce Chemical); ester nonionic surface-active agents such as Tween20, Tween 80 and Span 20 (all of them are trade names; manufactured byPierce Chemical); alcohol type nonionic surface-active agents such asNonidet P-40 (trade name; manufactured by Pierce Chemical); cholic acidtype nonionic surface-active agents such as CHAPS, CHAPSO, BIGCHAP andDEOXY-BIGCHAP (all of them are trade names; manufactured by PierceChemical); glycoside nonionic surface-active agents such ashexyl-β-D-glucopyranoside, octyl-β-D-glucopyranoside, octyl-β-glucoside,octyl-β-thioglucopyranoside and octylglucopyranoside; anionicsurface-active agents such as N-lauroyl sarcosine, sodium N-lauroylsarcosine, sodium laurylsulfate, lithium laurylsulfate and sodiumdodecylsulfate; and saponins such as saponin, Panax ginseng saponins,glycyrrhizin, glycyrrhizic acid and Bupleurum saponins. Among them,polyoxyethylene ether nonionic surface-active agents or alcohol typenonionic surface-active agents are preferably used. Particularly, TritonX-100 (trade name), Triton X-114 (trade name) or Nonidet P-40 (tradename) is preferred. Those agents for damaging the cell membrane may beused either alone or jointly in a mixed form.

[0127] In case where the agent capable of damaging cell membranes isapplied, a solution of said agent with a desired concentration can beapplied by dropping into a stationary culture medium containing cells orinto a stirred culture medium. Alternatively, a culture mediumcontaining cells, etc. can be added to a liquid containing said agent(capable of damaging cell membranes) in a predetermined concentrationeither gently or with stirring to form a mixture. In that case, additionmay be conducted with a tool such as pipette, injector (syringe),pipetter, dispenser, and spuit. In some cases, it is possible that thecells may be temporarily contacted with the agent capable of damagingcell membranes. Thus, for example, they may be just dipped in a solutioncontaining said agent (capable of damaging cell membranes). Preferably,such treatments can be conducted with an automated device equipped withmicroprocessors by adding a certain amount of a solution containing saidagent to a culture medium in accordance with a predetermined procedure.The device used therefor may be suitably selected from those which areknown in the art.

[0128] When cell membranes are damaged by a physical means, not onlycell membranes but also DNA, etc. in the cells may be damaged in somecases and, therefore, the treating condition is to be carefully selectedif necessary. For example, when ultrasonication is applied, it ispossible to treat the test sample by dipping a stirring rod thereinto orby placing it in a cup-like container equipped with a stirring rod. Itis also possible to use a continuously operable device. Such operabledevices used therefor may be suitably selected from those which areknown in the art. It is further possible to use an osmotic pressureshock method, a freezing and thawing method, etc. for damaging the cellmembrane.

[0129] When it is difficult to determine the cell viability directlyfrom the acquired data, a new calibration curve is prepared, ifnecessary, for each of the treating conditions so that the measuredvalue is corrected.

[0130] When the cell membrane of viable cells present in the test sampleafter the measurement of dead cells is subjected to a damaging treatmentto make the cells extinct, the fluorescent probe for nucleic acid alsoinvades into the cell which was once viable and is allowed to bond withnucleic acids and to emit fluorescence. Accordingly, the result is thatthe test sample has now the intensified fluorescence which gains by anamount of the fluorescence emitted by the cells which were once viable.When the intensified fluorescence intensity (Ft′) thereof is measured bya fluorometer, it is now possible to quantitatively determine the totalnumber of cells (i.e., total cell number) existing in the test sampleand then viable cell number can be determined by subtracting the deadcell number from the total cell number. To be more specific,

Ft=Ft′−Fb′

[0131] and

Fl=Ft−Fd=(Ft′−Fb′)−(Fd′−Fb)

[0132] are used for calculating the fluorescence intensity (Ft)corresponding to the total cell number and that (Fl) corresponding tothe viable cell number. From those data, the total number of cells andthe number of living cells, i.e. the number of viable cells, can bequantitatively determined by means of calibration curves, etc.

[0133] Fb′ is a background fluorescence intensity from the agent capableof damaging cell membranes, the fluorescent probe for nucleic acid, theculture medium and the target to be evaluated for cytotoxicity. When asurface-active agent is applied for damaging the cell membrane, thereare many cases where Fb=Fb′ and, in that case, Fl can be calculated by:

Fl=Ft′−Fd′

[0134] Several test samples under the same condition (i.e., in a singlecondition) are prepared and one half of the test samples is subjected toa determination for the above-mentioned dead cell numbers by theaforementioned technique while another half thereof is subjected to atreatment with a fluorescent probe for nucleic acid and a treatment ofdamaging the cell membrane whereby the total cell numbers are determinedby the same manner as in the case of the aforementioned method. When thedead cell numbers are subtracted from the total cell numbers calculatedas such, the viable cell numbers can be determined as well. In thismethod, it is possible to simultaneously conduct a step of determiningthe dead cell number and another step of determining the total cellnumber and, therefore, an efficient determination can be conducted.However, there is a possibility of resulting in the problems that it isnecessary to conduct the measurement for many test samples and that themeasurement error is big. On the other hand, when dead cell numbers arefirst determined and total cell numbers are then determined for the samesample, the time interval between the former and the latter measuringsteps causes a problem. Such a time interval may vary depending uponvarious conditions such as kinds of cell lines to be assayed,fluorescent probes for nucleic acid, agents capable of damaging cellmembranes, etc. and a short time is recommended, preferably within twohours, or more preferably within one hour. Accordingly, it is preferredthat various conditions such as kinds of cell lines to be assayed,fluorescent probes for nucleic acid, agents capable of damaging cellmembranes, etc. are taken into consideration to select the most suitabledetermination under the given condition.

[0135] The cell viability can be calculated from the total cell numberand viable cell number data obtained by the assay. When the dead cellnumbers are presumed to be nearly zero, then Fl≈Ft. Therefore, iffluorescence intensity is measured in such a manner that both atreatment with the fluorescent probe for nucleic acid and a treatmentwith the agent capable of damaging cell membranes are simultaneouslyconducted, it is possible to determine the total cell number or theviable cell number only by one step of fluorescence measurement. In thecase of pharmaceuticals, agricultural chemicals, cosmetics, foods, etc.,they are frequently assessed for their cytotoxicity, relying on data interms of viable cell number and cell viability. For example, there is acase where the concentration of drugs, etc. making 50% cells dead (i.e.IC₅₀) is calculated from the viable cell number data. In accordance withthe technique of the present invention, such a cytotoxicity can beconveniently and efficiently assessed.

[0136] In accordance with the present invention, it is possible todirectly determine the cell viability without counting the viable cellnumber.

[0137] The method according to the present invention can be conductedusing a measuring instrument having a function of outputting viable cellnumber and/or cell viability data by means of combination andsystematization of each operation in a series of the following steps:

[0138] first step: (i) measuring the fluorescence intensity of a testsample subjected to a treatment with a fluorescent probe for nucleicacid, capable of exclusively staining dead cells, and (ii) measuring thefluorescence intensity of a test sample subjected to not only (a) atreatment with said fluorescent probe for nucleic acid but also (b) atreatment of damaging cell membranes (this step includes each treatmentwhich is conducted prior to each measurement), and

[0139] second step: comparing both fluorescence intensities.

[0140] In said measuring instrument, it is preferred to conduct thepresent invention to utilize a system in which both a fluorescent probefor nucleic acid capable of exclusively staining dead cells and an agentcapable of damaging cell membranes are set for conducting each treatmentapplied prior to each measurement in the first step of said system. Insuch a measuring instrument, it is possible to apply a kit fordetermining the viable cell number and/or a kit for determining the cellviability and, accordingly, the determination of viable cell numbersand/or cell viability according to the present invention can beconducted efficiently. Embodiments of the apparatus (or system) whichconducts the assay according to the present invention in an automatedmanner include those as shown in FIGS. 1 and 2. In said system, a termreading “apparatus for determining viable cell number” is used but itmay also be understood as “apparatus for determining cell viability” or“apparatus for determining viable cell number and/or cell viability”. Inthe present specification and claims, said apparatus (or system) is tobe understood in a broad sense.

[0141]FIG. 1 is an embodiment of an apparatus for determining the viablecell numbers wherein the number of dead cells is first counted and thenthe total number of cells is counted for a single test sample inaccordance with the present invention. As such, said apparatus comprisesa spectrofluoro-photometer installed therein. Said apparatus comprises acuvette (for test sample) (6) equipped with a mixer, an excitationspectroscope (14), a fluorescence spectrophotometer (15), a light source(10), lenses (5, 9 and 11), polarizing plates (4 and 12) and aphotomultiplier (13). By referring to FIG. 1, (1) is an agent capable ofdamaging the cell membrane while (3) is a fluorescent probe for nucleicacid, capable of exclusively staining dead cells, and each of thereagents is set either in a tank or in a detachable container in theapparatus. The reagent which is set in such a tank or detachablecontainer can be injected using an injector (2) into the cuvette (6)equipped with a mixer. The apparatus is suitably equipped with a valveand a pump (not shown) for control. If necessary, it may also beoptionally equipped with a washing pump, a discharging pump, adirectional control valve, an air pump for stirring, a nozzle, etc. Inthe drawing, (7) is a transducer for transforming analog signals todigital ones and (8) is a control system. The cuvette (6) may besequentially moved upon necessity whereby plural cuvettes may beautomatically measured. It is also possible that the cuvette for testsample is kept in a constant-temperature bath. It is further possiblethat the cuvette for test sample (6) is immobilized while the injectorand the spectrofluorophotometer are sequentially moved if necessary toconduct an automatic measurement.

[0142]FIG. 2 illustrates an embodiment of an apparatus for determiningthe viable cell number for the case where several test samples under thesame condition are prepared, one half of the test samples is subjectedto a measurement for the aforementioned dead cell number by theaforementioned technique while another half thereof is subjected to atreatment with a fluorescent probe for nucleic acid and a treatment fordamaging the cell membrane whereby the dead cell number is measured forthe test sample to be assayed for the dead cell number in the samemanner as in the aforementioned technique followed by measurement of thetotal cell number. As such, said apparatus comprises aspectrofluorophotometer incorporated therein. Said apparatus comprises a96-well microplate (5) equipped with a plate mixer, a excitationspectroscope (15), a fluorescence spectrophotometer (16), a light source(11), lenses (7, 10 and 12), polarizing plates (6 and 13) and aphotomultiplier (14). In the drawing, (1) is a diluted suspension ofcells, which is set in a tank or detachable container in the apparatus.

[0143] In order to supply each well with a homogeneous suspension interms of cell density from the tank, a mixer is installed either in thetank or in the detachable container. (2) is an agent capable of damagingcell membrane and (4) is a dye for nucleic acid, capable of exclusivelystaining dead cells, and both said reagents are each set in a tank ordetachable container in the apparatus. The suspension or reagent set insuch a tank or detachable container can be distributed with a dispenser(3) into each well on a 96-well microplate (5) equipped with a platemixer. A disposable chip or nozzle is installed at the top end portionof the dispenser (3). Such a disposable chip or nozzle may be optionallyexchanged or washed if necessary. The top end of the dispenser (3) maybe branched whereby it is capable of distributing the reagent to two ormore sample wells simultaneously. In the drawing, (8) is a transducerfor transforming analog signals to digital ones and (9) is a controlsystem. Said microplates (5) may be made sequentially and continuouslymovable if necessary, so that automatic measurement for two or moremicroplates is possible. It is also possible that said microplates arefixed while the dispenser (3) and the spectrofluorophotometer aresequentially and continuously movable, so that automatic measurement canbe conducted.

[0144] In case where the present invention is conducted in such a mannerthat dead cell numbers are measured and then total cell numbers aremeasured for the same sample, it is efficient to conduct the measurementusing an apparatus as shown in FIG. 1.

[0145] Described hereinbelow is an example of “how to use” such anapparatus.

[0146] (I) A test sample such as a cell sample in a culture medium isset in a cuvette for test sample (6), equipped with a mixer, followed bystirring. A fluorescent probe for nucleic acid capable of exclusivelystaining dead cells (1) and an agent capable of damaging cell membrane(3) are set in a tank in the apparatus in advance.

[0147] (II) The fluorescent probe for nucleic acid capable ofexclusively staining dead cells (1) is distributed with a dispenser (2)into each cuvette (6) (for test sample) wherein the test sample is set.As a result, the test sample is stained. Thereafter, intensity offluorescence emitted therefrom is measured by a spectrofluorophotometer.The resultant measurement data are processed by converting analog signalforms to digital ones and transmitted to a control system (8). Theamount of the fluorescent probe (1) (for nucleic acid, capable ofexclusively staining dead cells) used is automatically controlled underbeing linked with a control system (8) for measuring the fluorescenceintensity.

[0148] (III) The agent (1) (of damaging cell membranes) is distributedwith the dispenser (2) into the cuvette (6) (for test sample) aftercompletion of the measurement process (II) with the result that allcells in the test sample are made dead. Thereafter, intensity of thefluorescence emitted therefrom is measured by a spectrofluorophotometer.The resultant measurement data are processed by converting analog signalforms into digital ones and transmitted to the control system (8). Thecell viability is determined by comparing with the data obtained in themeasurement process (II). If necessary, it is also possible that theabove-mentioned measurement is conducted for several samples wherein thecell number is known and appropriate to prepare a calibration curve forthis apparatus, so that the viable cell number, etc. is determined forthe samples where the cell number is unknown.

[0149] It is efficient to conduct the measurements using an apparatus asshown in FIG. 2 in the case where several test samples are preparedunder a single condition and one half of the test samples is subjectedto a dead cell number measurement by the above-mentioned method whileanother half is subjected to a treatment with a fluorescent probe fornucleic acid and to a treatment of damaging cell membrane whereby thetotal cell number is calculated in the same manner as in theabove-mentioned method.

[0150] Described hereinbelow is also an example of “how to use” such anapparatus.

[0151] (I) For preparing test samples under the same condition, the testsample is distributed as a diluted cell suspension (1) with a dispenser(3) into each well on a 96-well microplate (5) equipped with a platemixer. Thereafter, the microplate well is agitated with a plate mixer. Afluorescent probe (2) (for nucleic acid capable of exclusively stainingdead cells) and an agent (4) (capable of damaging cell membrane) are setin a tank in the apparatus in advance.

[0152] (II) The fluorescent probe for nucleic acid (2) (capable ofexclusively staining dead cells) is distributed with a dispenser into a96-well microplate (5) wherein the test sample is set. As a result, thetest sample is stained. Thereafter, intensity of the fluorescenceemitted from the sample in each well is measured by aspectrofluorophotometer. The obtained measurement data are processed byconverting analog signal forms to digital ones and transmitted to acontrol system (9). The amount of the fluorescent probe for nucleic acid(4) (capable of exclusively staining dead cells) is automaticallycontrolled in combination with the control system (9) for thefluorescence intensity measurement. Different dispensing nozzles foreach reagent are installed in the dispenser so that the reagent can bedistributed into plural wells by a single operation.

[0153] (III) Each of the agent (2) (capable of damaging cell membrane)and the fluorescent probe (4) (for nucleic acid capable of exclusivelystaining dead cells) is distributed with a dispenser into a 96-wellmicroplate (5) where the test samples under the same condition as thatused in the measurement (II) are set. As a result, all cells in the testsample are made extinct and simultaneously stained. Thereafter,intensity of the fluorescence emitted from the test sample in each wellis measured by a spectrofluoro-photometer. The obtained measurement dataare processed by converting analog signal forms to digital ones andtransmitted to the control system (9). Each amount of the agent (2)(capable of damaging cell membrane) and the fluorescent probe (4) (fornucleic acid capable of exclusively staining dead cells) isautomatically controlled in combination with the control system (9) forthe fluorescence intensity measurement. The measured analog signal dataare transformed into digital ones and communicated to the control system(9) and cell viability is determined by comparing with the data obtainedin the measurement process (II). If necessary, it is also possible thatthe above-mentioned measurement is conducted for several samples whereinthe cell number is known and appropriate to prepare a calibration curvefor this apparatus, so that the viable cell number, etc. is determinedfor the samples where the cell number is unknown.

[0154] Thus, the present invention provides an apparatus for determiningviable cell number and/or cell viability, characterized in that,

[0155] said apparatus is equipped with at least:

[0156] (a) a means for supplying a test sample with a fluorescent probefor nucleic acid (capable of exclusively staining dead cells),

[0157] (b) a means for measuring the fluorescence intensity of the testsample treated with said fluorescent probe for nucleic acid,

[0158] (c) a means enabling us to damage the cell membrane, and

[0159] (d) a means for measuring the fluorescence intensity of the testsample subjected to not only a treatment with a fluorescent probe fornucleic acid (capable of exclusively staining dead cells) but also atreatment of damaging the membrane and

[0160] said apparatus is further equipped with a means for comparingboth intensities of the above (b) and (d) for each of the fluorescenceintensities as measured.

[0161] Each of those constituting elements in said apparatus may be usedby selecting from those which have been known in the art, includingautomated apparatus for immunoassays and biochemical assays or may beused after suitable modifications thereto if necessary.

[0162] Preferred apparatus includes a system in which a computercontrolled by a program, such as a microcomputer, regulates one orplural means, or all of the above-mentioned means, i.e.,

[0163] (a) a means for supplying a test sample with a fluorescent probefor nucleic acid (capable of exclusively staining dead cells),

[0164] (b) a means for measuring the fluorescence intensity of the testsample treated with said fluorescent probe for nucleic acid,

[0165] (c) a means enabling us to damage the cell membrane, and

[0166] (d) a means for measuring the fluorescence intensity of the testsample subjected to not only a treatment with a fluorescent probe fornucleic acid (capable of exclusively staining dead cells) but also atreatment of damaging the membrane and

[0167] a means for comparing both intensities of the above (b) and (d)for each of the fluorescence intensities as measured.

[0168] In the method for determining viable cell number and/or cellviability and also the kit for determining viable cell number and/orcell viability in accordance with the present invention, it is possibleto jointly use each of various fluorescent probes for nucleic acid(capable of exclusively staining dead cells) as aforementioned, incombination with each of various agents (capable of damaging cellmembranes) as aforementioned. It is preferred that the aforementionedfluorescent probe for nucleic acid (capable of exclusively staining deadcells) is used in combination with any of the aforementioned varioussurface-active agents.

[0169] It is also possible to use a mixture prepared by mixing theaforementioned fluorescent probe for nucleic acid (capable ofexclusively staining dead cells) with any of the aforementioned varioussurface-active agents, if necessary in advance.

[0170] A preferred embodiment of the above-mentioned kit (for measuringthe viable cell number and/or cell viability) applicable to and suitablefor an apparatus for measuring the viable cell number (or apparatus formeasuring the viable cell number and/or cell viability) as shown in FIG.1 or FIG. 2 is a form wherein each of the fluorescent probes for nucleicacid (capable of exclusively staining dead cells) and the agents(capable of damaging cell membranes) is in advance charged in adetachable container for said apparatus. In that case, said fluorescentprobes and said agents (capable of damaging cell membranes) are eachcharged in different detachable containers but, if necessary, both maybe charged in the same detachable container.

EXAMPLES

[0171] Described below are examples of the present invention which areprovided only for illustrative purposes, and not to limit the scope ofthe present invention. It is to be understood that the present inventionis not limited to those examples but includes numerous embodiments.

Example 1 (Determination of Cell Viability)

[0172] (1) Method and Result

[0173] {circle over (1)} MOLT-4 cells (human acute lymphoblasticleukemia cells; received from Microbiological Laboratory of OsakaUniversity, Japan) were cultured on a Petri dish at 37° C. under a 100%humidified atmosphere of air containing 5% carbon dioxide. The culturemedium used was RPMI medium (No. 31800-071, manufactured by Gibco),supplemented with 10% fetal calf serum (hereinafter, referred to as“culture medium”).

[0174] {circle over (2)} When a cell density becomes high in the culturemedium, samples on a Petri dish were used as test samples fordetermining the cell viability. The test samples include (i) a sample ona Petri dish where the medium was appropriately changed (sample undergood culture condition) and (ii) two samples which were incubated on aPetri dish without changing the medium (samples (1) and (2) under badculture condition).

[0175] {circle over (3)} Each test sample was collected from the Petridish, placed in a cuvette for test sample and subjected to themeasurement described hereinbelow while agitating with a stirring rod.

[0176] Fluorescence intensity was measured by a spectrofluoro-photometer(Hitachi type F-4000, manufactured by Hitachi, Ltd., Japan) atexcitation wavelength: 420 nm and emission wavelength: 460 nm.

[0177] {circle over (4)} First, PO-PRO-1 Iodide (trade name; No. P-3581,manufactured by Molecular Probes) was added thereto (finalconcentration: 2.5 μM) whereupon Fd′ was measured. Thereafter, TritonX-100 (trade name; manufactured by Pierce Chemical) was added (finalconcentration: 0.05%) whereupon Ft′ was measured. Cell viability (orsurvival ratio of cells) was calculated from the above equation and theresults are shown in Table 1.

[0178] {circle over (5)} For comparison, cell viability was alsodetermined under a microscope for each sample by a Trypan Blue stainingtechnique and the results are shown in Table 1. The Trypan Blue stainingdata were nearly the same as those in Example 1. It has been found thatthe method according to the present invention is well applicable fordetermining the cell viability. It is also possible to conduct thismeasurement in an automated manner using an apparatus as shown inFIG. 1. TABLE 1 Cell Viability Method of this Staining with InventionTrypan Blue Sample under good 99.0% 98.3% culture condition Sample (1)under bad 96.0% 95.9% culture condition Sample (2) under bad 88.5% 84.8%culture condition

Example 2 (Quantitative Nature of Reaction between Cells and Stains forNucleic Acid)

[0179] (1) Method and Result

[0180] {circle over (1)} MOLT-4, HL-60 (human myelomonocytic leukemiacells), U937 (human histiocytic lymphoma cells) and HT-1080 (humanfibrosarcoma cells) were used as test samples. All of the cell linesother than MOLT-4 were purchased from Dainippon Pharmaceutical Co.,Ltd., Japan.

[0181] {circle over (2)} The test samples were diluted 1:1 with aculture medium in terms of cell concentration to give diluted cellsuspensions. The diluted cell suspension was plated in a 96-wellmicroplate at 180 μl per well.

[0182] {circle over (3)} To each well was added 10 μl of PO-PRO-1 Iodide(trade name) (final concentration: 2.5 μM), followed by addition of 10∥l of Triton X-100 (trade name) (final concentration: 0.05%). Themixture was agitated for about 30 seconds using a plate mixer, followedby fluorescence measurement. The results are shown in FIG. 3 and FIG. 4.

[0183]FIG. 3 shows that there is a proportional relationship between thenumber of floating cells and the fluorescence intensity. For HL-60 andU-937, there is a proportional relationship between the number offloating cells and the fluorescence intensity when cell numbers arewithin a range from 1.25×10⁴ to 4×10⁵ while, for MOLT-4, there is aproportional relationship between the number of floating cells and thefluorescence intensity when cell numbers are within a range from1.25×10⁴ to 2×10⁵.

[0184]FIG. 4 shows that there is a proportional relationship between thenumber of adhesive cells and the fluorescence intensity. For HT-1080,there is a proportional relationship between the number of adhesivecells and the fluorescence intensity when cell numbers are within arange from 300 to 1×10⁵.

[0185] Fluorescence intensity was measured with an MTP-32 CoronaMicroplate Reader (manufactured by Corona Denki K.K., Japan) atexcitation wavelength: 420 nm and emission wavelength: 460 nm. Thismeasurement can be also conducted in an automated manner using anapparatus as shown in FIG. 2.

[0186] (2) Discussion and Conclusion

[0187] From the results as shown in FIGS. 3 and 4, it has been foundthat, although floating cells and adhesive cells are to be subjected toa fluorescence intensity measurement at different measuring ranges,there is a proportional relationship between the number of cells and thefluorescence intensity. As a result, viable cell numbers can becorrectly determined by the method of the present invention.

Example 3 (Evaluation of Cytotoxicity by Drugs)

[0188] (1) Method and Result

[0189] {circle over (1)} Mitomycin C (anticancer agent; manufactured byKyowa Hakko Kogyo Co., Ltd., Japan) was diluted with a culture medium.The diluted drug was distributed into a 96-well microplate at 50 μl perwell.

[0190] {circle over (2)} Thereafter, MOLT-4 cells were plated in anamount of 3×10⁴ cells/150 μl per well.

[0191] {circle over (3)} Each well was incubated at 37° C. for two daysunder a 100% humidified atmosphere of air containing 5% carbon dioxideand subjected to the measurement described hereinbelow. In afluorescence measurement, an MTP-32 fluorometer (manufactured by CoronaDenki K.K., Japan) was used.

[0192] {circle over (4)} To each well was added 10 μl of PO-PRO-1 Iodide(trade name) (final concentration: 2.5 μM), the mixture was agitated forabout 30 seconds using a plate mixer and fluorescence was measured todetermine Fd′.

[0193] {circle over (5)} To each well was further added 10 μl of TritonX-100 (trade name) (final concentration: 0.05%), the mixture wasagitated for about 30 seconds using a plate mixer and fluorescence wasmeasured to determine Ft′.

[0194] {circle over (6)} Viable cell numbers and cell viability werecalculated from the aforementioned equation and the results are shown inTable 2.

[0195] {circle over (7)} For comparison, each sample was subjected to aviable cell number determination by an MTT assay system by referring toCancer Research, Vol. 47, 936-942 (1987) and the results are shown inTable 2.

[0196] Further, steps for determination of viable cell numbers by themethod according to the present invention and by the MTT assay aresummarized in Table 3. Similarly, the above-mentioned determinationaccording to the present invention can be conducted in an automatedmanner using an apparatus as shown in FIG. 2. TABLE 2 CytotoxicityAssessment of Mitomycin C Concentration of Mitomycin C Added (μg/ml)IC50 100 10 1 0.1 0 (μg/ml) (Method of the Present Invention) Viablecell numbers 3 24 63 76 100 2.2 (% control) Cell viability (%) 29 87 10099 98 (MTT Assay System) Viable cell numbers 3 34 95 120 100 5.5 (%control)

[0197] TABLE 3 Summary of Determination Steps for Viable Cell Number(Method of the Present Invention) {circle over (1)} PO-PRO-1 (50 μM): 10μl/well  1 minute {circle over (2)} agitation with a microplate mixer 30seconds {circle over (3)} measurement of fluorescence at 420/460 30seconds {circle over (4)} Triton X-100 (1%): 10 μl/well  1 minute{circle over (5)} agitation with a microplate mixer 30 seconds {circleover (6)} measurement of fluorescence at 420/460 30 seconds    Totaltime required  4 minutes (MTT Assay System) {circle over (1)}centrifugation at 2,000 rpm  5 minutes {circle over (2)} removal ofsupernatant liquid  2 minutes {circle over (3)} MTT (0.04%): 100 μl/well 1 minute {circle over (4)} incubation at 37° C., 5% CO₂-air  3 hours{circle over (5)} PBS: 100 μl/well  1 minute {circle over (6)}centrifugation at 2,000 rpm  5 minutes {circle over (7)} removal ofsupernatant liquid  2 minutes {circle over (8)} DMSO (100%): 150 μl/well 1 minute {circle over (9)} agitation with a microplate mixer 30 seconds{circle over (10)} measurement of absorbance at 550 nm 30 seconds   Total time required  3 hrs and 20 min

[0198] (2) Discussion and Conclusion

[0199] As shown in Table 2, the cytotoxicity of mitomycin C wasestimated by the method of the present invention to be IC50=2.2 μg/ml.When this value is compared with that (5.5 μg/ml) in the estimation byan MTT assay system which has been most commonly used for evaluation ofdrug cytotoxicity, it is within an order of μg/ml and has a differencewhich is permissible in the related art. It is therefore clear that themethod according to the present invention can be well applied forassessment of drug cytotoxicity. In addition, the method of the presentinvention has advantages that cell viabilities at various concentrationsof drugs can be calculated simultaneously and accordingly that manyinformation for evaluation of the drug is available. With regard to thetime required for the determination, the method of the present inventionfinishes within about five minutes as shown in Table 3 and, as comparedwith the known MTT assay system (which requires three hours and twentyminutes), it is now possible to reduce the necessary time significantly.Further, in view of the operability, the method of the present inventiondoes not need centrifugation, washing operation and dissolution of thedye. The instant technique is thus much more simple and convenient.

Example 4 (Damage to Cell Membrane by Ultrasonic Wave)

[0200] (1) Method and Result

[0201] {circle over (1)} A cultured medium (40 ml) containing 5×10⁶MOLT-4 cells was used as a test sample, distributed into a cuvette fortest sample and treated for 10 seconds with an ultrasonic pulse mastax(Sonicator Ultrasonic Processor Model W-225, Serial No. G6453, 20 kHz,Heat Systems-Ultrasonic Inc.) (Disruption intensity level: 3).

[0202] {circle over (2)} The ultrasonicated sample in each cuvette wassubjected to the measurement described hereinbelow while agitating witha stirring rod.

[0203] Fluorescence intensity was measured using aspectrofluoro-photometer (Model F-4000; Hitachi, Ltd., Japan) atexcitation wavelength: 420 nm and emission wavelength: 460 nm.

[0204] {circle over (4)} First, PO-PRO-1 Iodide (trade name; No. P-3581,manufactured by Molecular Probes) was added thereto (finalconcentration: 2.5 μM). Fluorescence intensity was measured and found tobe 410 (background fluorescence intensity: 10).

Applicability in Industry

[0205] In accordance with the present invention, fluorescent probes fornucleic acid which stain dead cells only (or capable of exclusivelystaining dead cells) and agents for damaging cell membranes (or capableof damaging cell membranes) are used whereby viable cell numbers and/orcell viability of many samples can be determined conveniently andquickly and, furthermore, correctly. Accordingly, the present inventioncan be applied to cytotoxicity evaluation for pharmaceutical drugs,agricultural chemicals, cosmetics, foods, etc. In addition, thecytotoxicity assessment can be conducted conveniently and quickly aswell.

What is claimed is

1. A method for determining viable cell number and/or cell viability,which comprises (i) measuring the fluorescence intensity of a testsample subjected to a treatment with a fluorescent probe for nucleicacid, capable of exclusively staining dead cells, (ii) measuring thefluorescence intensity of a test sample subjected to not only (a) atreatment with said fluorescent probe for nucleic acid but also (b) atreatment of damaging cell membranes, and (iii) comparing bothfluorescence intensities.
 2. The method according to claim 1 wherein (i)the test sample is treated with a fluorescent probe for nucleic acidcapable of exclusively staining dead cells, and the resultantfluorescence intensity is measured, and (ii) said test sample is thensubjected to a treatment for damaging cell membranes with the resultthat cells are destined to become extinct, thereby intensifying thefluorescence thereof and the resultant fluorescence intensity thereof ismeasured.
 3. The method according to claim 1 wherein (i) the test sampleis treated with a fluorescent probe for nucleic acid capable ofexclusively staining dead cells, and the resultant fluorescenceintensity of said test sample is measured, and (ii) another test sampleis subjected to not only (a) a treatment with said fluorescent probe fornucleic acid but also (b) a treatment of damaging cell membrane, and theresultant fluorescence intensity thereof is measured.
 4. The methodaccording to any of claims 1 to 3 wherein the fluorescent probe fornucleic acid is a cationic fluorescent stain or dye for nucleic acid. 5.The method according to any of claims 1 to 4 wherein the fluorescentprobe for nucleic acid is ethidium homodimer or a cyanine fluorescentdye capable of exclusively staining dead cells.
 6. The method accordingto any of claims 1 to 5 wherein the cell membrane is damaged with anagent capable of damaging cell membranes.
 7. The method according toclaim 6 wherein the agent capable of damaging cell membranes is asurface-active agent.
 8. The method according to claim 6 or 7 whereinthe agent capable of damaging cell membranes is a polyoxyethylenenonionic surface-active agent or alcohol nonionic surface-active agent.9. The method according to any of claims 1 to 8 wherein each operationin a series of the following steps is conducted by an automatedapparatus: (i) measuring the fluorescence intensity of a test samplesubjected to a treatment with a fluorescent probe for nucleic acid,capable of exclusively staining dead cells, (ii) measuring thefluorescence intensity of a test sample subjected to not only (a) atreatment with said fluorescent probe for nucleic acid but also (b) atreatment of damaging cell membranes, and (iii) comparing bothfluorescence intensities.
 10. A kit for determining viable cell numberand/or cell viability by the following steps: (i) measuring thefluorescence intensity of a test sample subjected to a treatment with afluorescent probe for nucleic acid, capable of exclusively staining deadcells, (ii) measuring the fluorescence intensity of a test samplesubjected to treatments with (a) said fluorescent probe for nucleic acidand (b) an agent capable of damaging cell membranes, and (iii) comparingboth fluorescence intensities, which comprises (A) said fluorescentprobe for nucleic acid, capable of exclusively staining dead cells, and(B) said agent capable of damaging cell membranes.
 11. An apparatus fordetermining viable cell number and/or cell viability, which comprises atleast (a) a means for supplying a test sample with a fluorescent probefor nucleic acid, capable of exclusively staining dead cells, (b) ameans for measuring the fluorescence intensity of the test sample whichis treated with said fluorescent probe for nucleic acid, (c) a meansenabling us to damage cell membrane, (d) a means for measuring thefluorescence intensity of the test sample which is subjected to not only(A) a treatment with said fluorescent probe for nucleic acid but also(B) a treatment of damaging cell membrane, and (e) a means for comparingthe fluorescence intensity (b) with that (d).
 12. The apparatusaccording to claim 11 wherein said apparatus is equipped with: (a) adevice for holding an agent capable of damaging cell membranes, (b) adevice for holding a fluorescent probe for nucleic acid capable ofexclusively staining dead cells, (c) an injector for drug capable ofsupplying the test sample with the agent from the above-mentioned device(a) or (b), (d) a container capable of holding the test sample therein,and (e) a spectrofluorophotometer.